Process for the manufacture of a high-purity activated factor VII concentrate essentially free of vitamin K-dependent factors and factors VIIIC and VIIICAg

Abstract

The invention relates to a process for the manufacture of a high-purity activated factor VII concentrate. This process comprises the use of a plasma free of cryoprecipitate, preferably of human origin, as well as at least one purification step involving chromatography at least once on an ion exchange resin, and a factor VII activation step, wherein the first stage is direct activation of the factor VII in the crude supernatant of plasma free of cryoprecipitate, without the addition of exogenous proteins. By virtue of the invention, the high-purity activated factor VII is essentially free of vitamin K-dependent factors and factors VIIIC and VIIICAg and has a factor VIIa/factor VII ratio greater than 5 with a specific activity of the activated factor VII greater than 200 IU/mg of proteins.

What is claimed is: 
     
       1. A process for the manufacture of a high purity concentrate containing activated factor VIIa, comprising: a) providing a plasma containing factor VII and free of cyroprecipitate;   b) directly activating the factor VII in the plasma without the addition of exogenous proteins and without the precipitation of lipoproteins, to obtain a plasma free of cryoprecipitate containing an increased amount of activated factor VIIa, said activating comprising contacting said plasma with an amount of pulverulent dextran sulfate, insoluble in said plasma, sufficient to activate said factor VII without precipitation of lipoproteins; and   c) purifying said plasma containing activated factor VIIa and non-activated factor VII by chromatography with an ion exchange resin.   
     
     
       2. A process for the manufacture of a high purity concentrate containing activated factor VIIa, comprising: a) providing a plasma containing factor VII and free of cryoprecipitate;   b) directly activating the factor VII in the plasma without the addition of exogenous proteins and without the precipitation of lipoproteins, to obtain a plasma free of cryoprecipitate containing an increased amount of activated factor VIIa, said activating comprising contacting said plasma with pulverulent dextran sulfate, insoluble in said plasma, in an amount of about 0.1 g per liter of plasma; and   c) purifying said plasma containing activated factor VIIa and non-activated factor VII by chromatography with an ion exchange resin.   
     
     
       3. A process according to claim 1 or 2 wherein the dextran sulfate is in the form of beads. 
     
     
       4. A process according to claims 1 or 2 wherein the direct activation of factor VII is carried out at a temperature of between about 0° C. and about +4° C. 
     
     
       5. A process according to one of claims 1 or 2 wherein the direct activation is followed by anion exchange chromatography with a DEAE anion exchange resin on the cryoprecipitate-free plasma containing the activated factor VIIa and non-activated factor VII, so as selectively to adsorb all vitamin K-dependent factors, including activated factor VII and non-activated factor VII, present in the plasma. 
     
     
       6. A process according to claim 5 wherein the vitamin K-dependent factors adsorbed on the DEAE ion exchange column are non-selectively desorbed into a first eluate. 
     
     
       7. A process according to claim 6 wherein the first eluate containing the vitamin K-dependent factors, is passed over an anion exchange resin of the quaternary amine type, so as selectively to adsorb vitamin K-dependent factors present in the first eluate. 
     
     
       8. A process according to claim 5, wherein said anion exchange resin of the quaternary amine type is in the form of agarose crosslinked to the extent of about 6%. 
     
     
       9. A process according to claim 8, wherein the crosslinked agarose comprises a small C 1  -C 6  spacer arm bearing said quaternary amine. 
     
     
       10. A process according to claim 7 additionally comprising selective desorption of the activated factor VIIa and the non-activated factor VII by elution of the quaternary amine resin with a selective desorption buffer solution into a second eluate. 
     
     
       11. A process according to claim 10 wherein the selective desorption buffer comprises 4 mM trisodium citrate and 250 mM sodium chloride, and has a pH of about 6. 
     
     
       12. A process according to claim 10 wherein the second eluate, which contains activated factor VIIa and non-activated factor VII in aqueous form and is substantially free of other vitamin K-dependent factors, is then subjected to a viral inactivation step. 
     
     
       13. A process according to claim 12, wherein the viral inactivation step comprises treating the selective desorption buffer eluate with a TNBP/Tween mixture comprising 0.3% TNBP with respect to the total factor VII present and 1% Tween with respect to the total factor VII present, passing the treated eluate over a quaternary amine column with a buffer permitting selective adsorption, and selectively desorbing the factor VII from the column with a selective desorption buffer. 
     
     
       14. A process according to claim 13, wherein the selective adsorption buffer comprises 150 mM NaCl and 4 mM sodium citrate, and has a pH of about 6. 
     
     
       15. A process according to claim 12 wherein the virally inactivated second eluate is subjected to diafiltration against a second buffer to form a diafiltration product. 
     
     
       16. A process according to claim 15, wherein the second buffer is compatible with injection in humans, comprises 100 mM NaCl and 4 mM trisodium citrate, and has a pH of about 7. 
     
     
       17. A process according to claim 16, wherein said second buffer also comprises 3 g/l lysine. 
     
     
       18. A process according to claim 15 wherein the product of diafiltration is concentrated to 5000 IU of activated factor VIIIa per 20 ml and filtered under sterile conditions on a sterilizing filter with a pore size of 0.2 micrometer. 
     
     
       19. A process according to claim 18, additionally comprising lyophilizing the filtered product. 
     
     
       20. A process according to claim 1 or 2, wherein the concentrate has a factor VIIa/factor VII ratio greater than 5. 
     
     
       21. A process according to claim 20, wherein said ratio is greater than 7. 
     
     
       22. A process according to claim 21, wherein said ratio is about 10. 
     
     
       23. A process according to claim 1 or 2, wherein the plasma is of human origin.